The present invention relates to a three-dimensional NMR (nuclear magnetic resonance) spectroscopy.
FIG. 1 shows a one-dimensional (1D) NMR spectrum of oxytocin. This spectrum contains various kinds of information including intensities, chemical shifts, coupling constants, multiplicity, and line widths, and these kinds of information are mixed together in a quite complex manner. In order to separate these kinds of information, complicated spectral analysis is needed.
Two-dimensional (2D) NMR spectroscopies have been evolved to solve the above-described problem with the 1D NMR spectroscopy. Generally, a 2D NMR spectroscopy yields a higher resolution and makes it easier to analyze spectra than the prior art method. Further, the 2D spectroscopy enables elucidation of nuclear spin-spin interactions, and offers other advantages.
The 2D NMR spectroscopy which was first introduced was correlation spectroscopy (COSY) using two 90.degree.-pulses. Today, the concept of 2D NMR spectroscopy has been extended to NOE spectroscopy (NOESY), multiple-quantum filtering-correlation spectroscopy (MQF-COSY), spin echo spectroscopy (SECSY), H-C COSY, and other 2D techniques using three or four pulses. FIG. 2 shows the manner in which these 2D NMR techniques have evolved.
FIG. 3 shows pulse sequences used in these 2D NMR techniques. FIG. 3(a) shows a pulse sequence used for COSY. FIG. 3(b) shows a pulse sequence used for SECSY. FIG. 3(c) shows a pulse sequence used for MQF-COSY. FIG. 3(d) shows a pulse sequence used for NOESY. When homonuclear species are observed, the number of pulses of such a pulse sequence can be increased up to four.
A typical process of measurement performed by 2D NMR spectroscopy is next described by referring to FIG. 4(a), where a pulse sequence using three 90.degree.-pulses is employed. A first experiment consists of four periods, i.e., a preparation period of .tau..sub.p for maintaining the nuclear magnetization in its appropriate initial condition before the application of a first 90.degree.-pulse, an evolution period of t.sub.1 between the first 90.degree.-pulse and a second 90.degree.-pulse, a fixed mixing period of .tau..sub.m between the second 90.degree.-pulse and a third 90.degree.-pulse and a detection period of t.sub.2 subsequent to the third 90.degree.-pulse, or detection pulse. The phase and the amplitude of a free induction decay (FID) signal which is detected during the detection period of t.sub.2 reflects the behavior of the magnetization in the evolution period t.sub.1, which is separated from the detection period of t.sub.2 by the mixing period of .tau..sub.m.
The obtained data is given by S (t.sub.1, t.sub.2), where t.sub.1 and t.sub.2 are variables. The data is expressed in the form of a two-dimensional matrix. The nuclear magnetization evolves at resonant frequencies .omega..sup.(1).sub.rs and .omega..sup.(2).sub.tu during the period t.sub.1 and t.sub.2, respectively. Information about the magnetization existing during t.sub.1 is coupled to information about the magnetization existing during t.sub.2, by the pulse applied during the mixing period. The degree of coupling depends upon the pulse or pulse train constituting the mixing period. The action of the pulse or pulse train is mathematically expressed by various rotation operators R. The matrix elements R.sub.rs,.sub.tu of such a rotation operator represent intensities at positions .omega..sup.(1).sub.rs, .omega..sup.(2).sub.tu in a 2D spectrum. This is shown in FIG. 4(b), where the vertical and the horizontal axes express .omega..sub.1 and .omega..sub.2, respectively.
When this 2D NMR spectroscopy is employed, a measurement must be repeated many times with different values of t.sub.1. Since the accumulation technique that is conventionally used to enhance the signal-to-noise ratio is also utilized, the time actually taken for a series of measurements reaches several hours to tens of hours.
The present situation is that where organic substances are investigated by 2D NMR spectroscopy, almost all of the aforementioned 2D NMR techniques are utilized to make the spectral assignment. Therefore, a different kind of pulse train must be used to obtain a different kind of information. Of course, these different kinds of measurements are carried out at different instants of time, even on different days. Consequently, it is inevitable that the observational environment, including the conditions of the NMR spectrometer used, varies among individual measurements. Also, when an unstable substance undergoes investigation, the substance may decompose while a series of measurements is being conducted. In this way, it is impossible to obtain every 2D NMR spectrum from the sample under the same conditions. For this reason, the results of analysis derived by combining various 2D NMR spectra involve error or some degree of ambiguity.